1. Technical Field
This invention relates generally to the field of analysis of cells and particles. In particular, the invention provides impedance based apparatuses, microplates and methods for analyzing cells and particles. The present apparatuses, microplates and methods can be used in monitoring cell or particle attachment, migration and invasion. The present apparatuses, microplates and methods can also be used in identifying modulators of cell or particle attachment, migration and growth.
2. Background Art
Growth and metastasis, or pathological cell migration, are the fundamental properties of malignant cells. Better understanding of molecular mechanisms underlying the pathological cell migration will improve cancer treatment and prevention.
To dissect the mechanisms, research models both in vivo and in vitro have been used. In vivo models can monitor an entire process of cancer metastasis in experimental animals, but are not suitable for large scale evaluation of the properties of malignant cells. In in vitro models, cancer cell growth and migration activities can be more quantitatively analyzed, including transmigration of cells through a monolayer or multi-layers of cultivated cells or explanted tissues, growth of cells through natural and artificial extracellular matrixes or similar cellular structures, and cell motility in response to chemotactic agents.
Currently, there are three common methods available for detecting cell growth and/or cell migration in vitro, which include:
(1) Chemoattractant-induced migration (i.e. chemotaxis, which is the directional response of biological cells or organisms to concentration gradients of chemicals) /invasion and trans-well migration assay (Falk, W., “A 48 Well Micro Chemotaxis Assembly for Rapid and Accurate Measurement of Leukocyte Migration.” Journal of Immunological Methods, Vol: 33, pages, 239-247, 1980; Richards K. L. and J. McCullough, “A Modified Microchamber Method for Chemotaxis and Chemokinesis.” Immunological Communications, Vol: 13, pages: 49-62, 1984): cells in inserts invade and migrate across a layer of an artificial pored membrane (polyvinylprrolidone-free polycarbonate membrane or polyethylene terephthalate membrane) into lower chambers containing a given chemoattractant. The migrated cells that are usually attached on the other side of the membrane are then labeled with either chemical dyes (Neuroprobe, Inc., See: Neuroprobe Inc. www. Neuroprobe.com/protocol/pt—96a.html) or fluorescent dyes, followed by cell counting under the microscope or by a spectrofluorometer (TECAN, Coster, BD Biosciences, (Ilsley, S. R. 1996. MATRIGEL® Basement Membrane Cell Invasion Chamber. Becton Dickinson Technical Bulletin #422; www.bdbiosciences.com/discovery_labware/technical_resources/techbulletins.html; BD BioCoat™ FluoroBlok™ Tumor Cell Invasion System, www.bdbiosciences.com/discovery_labware/Products/drug_discovery/insert_systems/fluoroblok_invasion/; TECAN. www.tecan.com/migration_introl.pdf). For example, U.S. Pat. No. 5,284,753 discloses a multiple-site chemotactic test apparatus and method. In one approach, chemotactic factors and controls are placed at preselected areas on the top surface of a bottom plate, while a membrane filter containing pores of appropriate sizes and topped with cell suspensions is placed above the bottom plate so that the drops of chemotactic factors and controls contact the filter membrane directly below the locations of the cell suspensions. The cells that migrate through the membrane filter under influence of chemotactic factors and controls for a period of time are counted and determined.
(2) In situ cell diffusion and migration assay: Cells are spotted onto a chemoattractant-coated glass slide. As cells migrate and diffuse from the original spotted positions, the diameter of the spot where cells are present increases. The diameter of the spot is measured after incubation. The migration is determined based on the size of the cell spot over incubation time (Berens, M. E., et al, “The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay”, Clinical and Experimental Metastasis. Vol: 12, pages: 405-415,1994; Creative Scientific Methods. www.cre8ive-sci.com/process.html).
(3) In vitro wound healing assay: Scratching cells off a cell monolayer and measuring the healing process of scratch under a microscope (Miyata K., et al. “New wound-healing model using cultured corneal endothelial cells. 1. Quantitative study of healing process”, Jpn J Ophthalmol. Vol: 34, pages: 257-266, 1990).
In situ cell diffusion and in vitro wound healing assays are easy to perform and costs for these assays are low. However, since neither of them is able to differentiate cell migration from cell proliferation, the results obtained from these two assay systems are not very reliable. The trans-well invasion and migration assay is the most commonly used and well-accepted in vitro method for analyzing cell invasion and migration. Such an assay uses an apparatus that contains an insert that forms an upper chamber, and a lower chamber. The two chambers are separated with an artificial extracelluar matrix (ECM)-coated pored membrane (or called membrane filter). Invasive cells placed in inserts or upper chambers can invade and migrate across an artificial ECM-coated microporous membrane to lower chambers. The invasion and migration activity is determined by counting the cells that have passed through the ECM-coated membrane into the lower chamber. This in vitro assay system appears to be able to mimic in vivo cell invasion and migration process, and to be suitable for large-scale assays for investigating responsive genes and intracellular signal transduction pathways. However, data collecting methods or technologies currently available for the in vitro system are either dye-based or radioactive isotope-based methods, and in the most cases, manual counting is required for data acquisition. Counting the cells that invade and migrate through the membrane to the lower chamber can be difficult and time consuming, which limits the assay accuracy and throughput significantly. Problems with sensitivity, reproducibility, and simplicity are often encountered when such data acquisition methods are used. A more efficient in vitro assay system is highly demanded nowadays, given that the identification of molecules and intracellular signal transduction pathways involving cell invasion and migration is increasing rapidly. To improve the assay accuracy and throughput, two new systems have been introduced by Amersham Pharmacia and Becton-Dickson (BD) Biosciences. The Amersham system uses a scintillation microplates (cytostar-T) (Graves, R., et al, “A novel assay for cell invasion using Cytostra-T scintillating microplates”. Scientific poster,http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/DrugScr+Scientific+Posters) to measure the cell invasion and migration activity using [14C] and [35S] labeled cells. In the test wells, a lower layer of ECM gel is added to form a barrier preventing the labeled cells from reaching the scintillant containing baseplate. The labeled cells are then added in an upper layer of ECM gel, and the microplate is incubated overnight. Only cells invading the lower layer of ECM gel and gaining proximity to the scintillant generate a signal in the assay. The system allows for automation and real time monitoring of cells that have penetrated the ECM gel. However, the requirement for radioactive isotope labeling limits the use of the system. The system developed in BD Biosciences uses a light-tight FluoroBlok PET membrane that is specifically designed to block the transmission of light from 490 to 700 nm (BD BioCoat™ FluoroBlok™ Tumor Cell Invasion System: http://www.bdbiosciences.com/discovery_labware/Products/drug_discovery/insert_systems/fluoroblok_invasion/). The testing cells should be first stained with a fluorescent dye and than placed into the light-tight FluoroBlok PET membrane inserts. Invaded cells on the reverse sides of the inserts can be monitored by a fluorescent detector in real time without destroying the assays. Using this assay system the invasion assay productivity and throughput are significantly improved. However, since not every cell type can be homogenously labeled by the fluorescent dye and labeling has in some cases been found to alter cell invasion and migration, the application of the system has also been significantly limited.
Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomaterials with electronic devices. There has been a growing interest in applying electronic methods for cell manipulation and cell analysis.
Cell-substrate impedance measurement is an electronic method for cell monitoring and sensing. Adherent cells are cultured on the surface of microelectrode structures located on a solid substrate. The presence and absence of cells on the electrode surface affect sensitively the electronic and ionic passage between cell culture media and the electrode structures (see, for example, Giaever I. and Keese C. R., “Monitoring fibroblast behavior in tissue culture with an applied electric field”, Proc. Natl. Acad. Sci. (USA), 1984, vol. 81, pp 3761-3764). Thus, interrogating the electrode impedance provides important information about biological status of the cells present on the electrodes. U.S. Pat. No. 5,187,096 discloses a cell substrate electrical impedance sensor with multiple electrode arrays. Each electrode pair within the impedance sensor for measuring the cell-substrate impedance comprises one small electrode (a measuring electrode) and one large electrode (a reference electrode) on two different layers. The difference between the electrode sizes ensures that the measured impedance change relative to the impedance when no cells are present on the electrodes is directly correlated with the cell numbers and sizes, generally 20-50 cells, or even single cells attached to or grown on the measuring electrodes. Some applications of the cell sensor include the monitoring of conditions within bioreactors, within cell cultures, the testing of compounds for cytotoxicity, research of cell biology to detect cell motility, metabolic activity, cell attachment and spreading, etc. However, this impedance sensor with two layered structures is somewhat complicated with the measuring electrodes on one layer and the reference electrodes on another layer. The selected electrode area for the small electrodes limits the maximum of 50 cells being monitored.
U.S. Pat. No. 4,686,190 disclosed a device for in vitro study of cell migration across a monolayer of epithelia cells while simultaneously measuring the transepithelial electrical resistance of the epithelia monolayer.
WO 02/42766 A2 disclosed a device and method for investigating the effects of chemical and other factors on cell movement. In the dveice, cells migrate in an under-agarose environment and their position is monitored using a system capable of measuring changes in impedance and other electrical parameters of the system at a target electrode lithographed onto a substrate as the cells arrive at target.
Other data collecting methods or technologies currently used for in vitro cell migration assays are typically dye-based, and in the most cases manual counting is required. Problems with sensitivity, reproducibility, and simplicity are often encountered when the dye-based procedures are used.
To overcome the limitations of the existing art, the invention aims to expand the usage and application of electrical field and other electronic methods for measuring and analyzing cells, non-cell particles, and biological, physiological, and pathological conditions of cells or non-cell particles. To this end, the invention provides innovative cell migration assay devices using microelectronic technology.